Decellularisation of matrices

ABSTRACT

A method of preparing matrices or tissue engineered biomaterials for implantation, and in particular to a method of improving decellularisation of matrices or tissue engineered biomaterials prior to implantation. The method employs a single anionic detergent in combination with protease inhibitors.

[0001] The present invention relates to a method of preparing matricesor tissue engineered biomaterials for implantation, and in particular toa method of improving decellularisation of matrices or tissue engineeredbiomaterials prior to implantation.

BACKGROUND TO THE INVENTION

[0002] A large variety of body implants are known for medical uses suchas substitute vascular prostheses, skin dressings and coverings, and forother purposes. The implant material can be synthetic or body tissuesfrom the same species or other species as the species to be implanted.When body tissues and structures are to be implanted, they may be usedfresh from the donor but in many cases, it is preferred to have somemeans of preserving the implant tissue for later use. There are severalpreservation techniques currently available including cryopreservationand chemical fixation with cross-linking agents such as glutaraldehyde,polyglycidyl ether and carbodiimide. In order to prepare the implanttissue for later use it is desirable to decellularise the tissue priorto storage whilst minimising any damage to the physical structure of thetissue matrix itself. This decellularisation can be important inimproving the biocompatability and reducing the immunological reactionin the tissue graft.

[0003] It is known from the prior art to use anionic detergents such assodium dodecyl sulphate (SDS) for the extraction of cellular components.SDS extraction was first described in U.S. Pat. No. 4,323,358 as amethod of preventing or delaying the calcification ofglutaraldehyde-fixed Hancock heart valve bioprosthesis, the method isreferred to as the “Hancock T6 treatment”. In this method, fixed tissueis contacted with SDS so as to retard calcification. However, seriouslimitations of the method have been reported (Bodnar E et al, Thorac.Cardiovasc. Surgeon.1985 34: 82-85; Courtman D Wet al, J Biomed MaterRes. 1994 28: 655-666; Wilson G J et al. ASAIO Trans 1990 36: M340-343).These researchers all report that SDS has a deleterious effect on heartvalve extracellular matrix (ECM) and in particular on the collagen andelastin fibre components.

[0004] In order to mitigate the effects of SDS on ECM, U.S. Pat. No.4,776,853 describes the use of an earlier pre-treatment with othernon-ionic detergents, such as Triton X-100 so that SDS is only employedas the second detergent in a multistage detergent decellularisationprogram.

[0005] A further problem associated with decellularising tissue implantis to minimise the degradation to the ECM during the process. It isknown to use protease inhibitors to prevent such degradation duringincubation with a non-ionic detergent in the first stage of themultistage detergent decellularisation program and also to use them toprevent naturally occurring proteases from attacking collagens. Thereare a number of different proteases that reside within the tissuematrix, either in direct association with the cells themselves or boundwithin the ECM. One of the largest of the protease families, the matrixmetalloproteases (MMPs), has a wide range of substrate specificitiesincluding collagen, laminin, fibronectin and elastin. Another importantfamily of matrix-degrading proteases are the plasminogen activators,which generates the broad specificity protease plasmin from the abundantzymogen plasminogen. As well as proteolytic activity, plasmin has thefurther ability of activating members of the MMP family. However, mostof the protease inhibitors are inherently toxic which is undesirable ifthe matrix is to be seeded with living cells and implanted into a humanor animal. Moreover, some of the protease inhibitors used so far, forexample PMSF, are extremely unstable in solution having a half life ofless than 1 hour, and since decellularisation is a lengthy process i.e.several days, this limits the choice of inhibitors that have sufficienthalf lives.

[0006] A method which could simplify the decellularisation processwhilst minimising damage to ECM would offer significant advantage overcurrent practices.

STATEMENT OF THE INVENTION

[0007] In its broadest aspect, the present invention provides a methodof decellularising a tissue matrix using an anionic detergent at aconcentration sufficient to effect decellularisation but at aconcentration which maintains the histoarchitecture of the ECM, the soleanionic detergent being used in conjunction with protease inhibitors.

[0008] According to a first aspect of the invention there is provided amethod of preparing biological material for implantation comprising thesteps of:

[0009] (i) incubating the biological material with a buffer solution ata mild alkaline pH which includes active amounts of a proteolyticinhibitor;

[0010] (ii) incubating the biological material with an anionic detergentat a mild alkaline pH at a concentration which is sufficient to effectdecellularisation but which maintains the histoarchitecture of thebiological material;

[0011] (iii) washing the biological material with a buffer solution at amild alkaline pH both with and without active amounts of proteolyticinhibitors;

[0012] (iv) incubating the biological material with one or more enzymesselected from the group comprising DNase Type I, DNase Type II, and/orRnase and optionally;

[0013] (v) placing the biological material in a cryoprotectant medium.

[0014] Preferably the method does not include any additional detergentincubation steps.

[0015] Reference herein to decellularisation is intended to include theremoval of cellular membranes, nucleic acids, lipids, cytoplasmiccomponents and retaining an ECM having as major components collagens andelastins.

[0016] Preferably, the buffer solution is hypotonic or isotonic. It willbe appreciated that each may be used either as the sole buffer or incombination at different stages of the method and that use of hypotonicor isotonic buffer is not intended to limit the scope of the presentapplication.

[0017] The method may include the further step of cryopreserving thebiological material in a cryogen such as liquid nitrogen until required.

[0018] Preferably, the proteolytic inhibitors are ethylene diaminetetraacetic acid (EDTA) and Aprotinin.

[0019] We have found Aprotinin particularly effective as a proteolyticinhibitor and of particular utility because of its low toxicity,stability in solution at different pHs and stability at a variety ofdifferent temperatures.

[0020] Typically, EDTA is used at a concentration range of 1 to 100 mMor 0.01-1.0% (w/v) and typically at 10 mM or 0.1% and Aprotinin at aconcentration range of 1-100 KIU and typically at 10 KIU.

[0021] Preferably, the mild alkaline conditions of step (i) are in therange of pH above 7.0 and up to pH 10.0, and more preferably are at pH8.0.

[0022] Preferably, the incubation period of step (i) of the method isfor between 8 to 20 hours and more preferably is for 14 hours.

[0023] Preferably, the anionic detergent is sodium dodecyl sulphate(SDS) or sodium deoxycholate.

[0024] Preferably, SDS is used at a concentration equal to or below 0.1%(w/v), and equal to or above 0.03% (w/v).

[0025] Reference herein to the term % (w/v) refers to the percentage inweight (grams) per unit volume (100 ml), thus 0.1% w/v is equivalent to0.1 gm dissolved in 100 ml.

[0026] Prior art methods in which SDS has been suggested fordecellularisation use concentrations of SDS equal to or greater than 1%(w/v) in order to effect decelluarisation. We have found that usinganionic detergents at this concentration results in destabilisation ofprotein interactions and/or solubilisation thus leading to degradationof ECM proteins. It has been the prevailing wisdom that SDS would not beeffective below a concentration of 1% (w/v). However, our results haveshown surprisingly, that a concentration of 0.1% or below it iseffective for decellularisation when conducted in the presence ofprotease inhibitors and that there is no damage to the ECM.

[0027] Our further studies using a second non-ionic detergent such asTriton X-100, n-hexyl-β-D-glucopyranoside, TWEEN 20 and MEGA 10 and thezwitterionic detergent CHAPS showed no effect on the decellularisationproduced by a low concentration of SDS alone. The results showed thatuse of a second detergent had no significant effect on decellularisationof porcine heart valves even after a period of 72 hrs. Thus, the presentinvention is of particular advantage in that we have shown that there isno requirement for a second non-ionic detergent. Accordingly, thepresent invention has obviated the need for a multistage detergentprocess.

[0028] Preferably, sodium deoxycholate is used at a concentration equalto or below 2.0% (w/v) and equal to or above 0.5% (w/v).

[0029] Preferably, the incubation period of step (ii) of the method isfor between 20 to 28 hours and more preferably is for 24 hours.

[0030] Preferably, the mild alkaline conditions of step (ii) are in therange of pH above 7.0 and up to pH 10.0, and more preferably are at pH8.0.

[0031] Preferably, the washing step (iii) of the method involvesmultiple washes, typically ×3 washes with tris buffered saline(preferably 0.15M NaCl, 0.05M tris in distilled water) containingprotease inhibitors (0.1% EDTA and 10 KIU/ml Aprotinin), and further,multiple washes, typically ×3 washes with tris buffered saline withoutthe protease inhibitors.

[0032] Preferably, the mild alkaline conditions of step (iii) are in therange of pH above 7.0 and up to pH 10.0, and more preferably are at pH8.0.

[0033] Preferably, the incubation step (iv) of the method is for 4-6hours at 37° C.

[0034] The DNase Type I, DNase Type II or Rnase are employed as lowionic strength solutions in an amount effective so as to eliminatenucleic acids and provide a tissue matrix of limited immunogenicity.Accordingly any other agents which are capable of the same function areincluded within the scope of the present invention.

[0035] Preferably, DNAse I is used at a concentration range of 5.0-100%g/ml and typically at 20 μg/ml and RNAse A at a concentration range of0.1-10 μg/ml and typically at 1 μg/ml.

[0036] Preferably, the biological tissue is prepared in step (v) of themethod for storage by placement in a cryoprotectant, such as and withoutlimitation, Dulbecco's modified eagles medium (DMEM) containing 10%(v/v) foetal bovine serum (FBS) and 10% (v/v) dimethyl sulphoxide(DMSO).

[0037] According to a further aspect of the invention there is provideda tissue implant comprising ECM from which have been removed cellularmembranes, nucleic acids and other cytoplasmic components the tissueimplant having been produced by the method of the present invention.

[0038] In summary, preferred embodiments of the present inventionprovides a method of decellularisation of a tissue matrix which resultsin no significant deleterious effects to the ECM proteins and subsequenthistoarchitecture of the aortic valve, as assessed by both histologicaland biomechanical techniques and wherein a single anionic detergent suchas SDS or sodium deoxycholate is used at a concentration sufficient tocause decellularisation;

[0039] as a single stage detergent;

[0040] at low concentrations that effect decellularisation whilstmaintaining the ECM in good condition;

[0041] in combination with the protease inhibitors EDTA (as an inhibitorof MMPs) and Aprotinin (inhibitor of serine family of plasminogenactivators);

[0042] for a period of about 24 hours;

[0043] According to a yet further aspect of the invention there isprovided a product comprising a combination of an anionic detergent at aconcentration hereinbefore described and a proteolytic inhibitor ashereinbefore described and optionally including a set of instructionsfor use thereof for use in the method of the present invention.

[0044] Alternatively the product comprising may comprise concentratesfor dilution and use in a method.

[0045] The present invention will now be described by way of exampleonly with reference to the following Figures wherein:

[0046]FIG. 1A illustrates a cross section of heart valve leaflet treatedwith 0.05% SDS solution at ×10 magnification;

[0047]FIG. 1B illustrates FIG. 1A at ×40 magnification;

[0048]FIG. 2A illustrates a cross section of heart valve leaflet treatedwith 0.02% SDS solution at ×10 magnification;

[0049]FIG. 2B illustrates FIG. 2A at ×40 magnification;

[0050]FIG. 3A illustrates a photomicrograph at ×400 magnification offresh porcine patellar tendon stained with heamatoxylin and eosin and;

[0051]FIG. 3B illustrates a photomicrograph at ×400 magnification ofporcine patellar tendon following the de-cellularisation treatmentaccording to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0052] With reference to FIGS. 1A and 1B there is shown a cross sectionof a heart valve leaflet treated with concentrations of 0.03% SDS. Totalleaflet decellularisation was observed at this concentration. However,at concentrations below 0.05% (FIGS. 2A and 2B), whole cells or cellfragments were found to have been retained by the matrix. Cross sectionof a heart valve leaflet treated with 0.02% SDS is therefore aconcentration below which decellularisation does not occur. It can beseen that cell fragments and whole cells have been retained within thematrix (Blue/black pigment). Following the method of the presentinvention, patella tendons may also be successfully decellularised (FIG.3B).

EXAMPLE 1 Porcine Aortic Valves

[0053] Porcine hearts were procured from a local abattoir within 2 hoursof slaughter and transported on ice to the laboratory. On arrival at thelaboratory, aortic valve roots were dissected from the heart and washedin transport solution [Hanks' balanced salt solution (HBSS), 10 KIU/mlAprotinin, 10 u/ml penicillin, 100 μg/ml streptomycin, 100 U/mlNystatin, 10 mM HEPES pH7.6). The aortic valves were incubated overnight(14 hours) in hypotonic tris buffer (10 mM tris pH8, 0.1% (w/v) ethylenediamine tetraacetic acid (tDTA), 10 KIU Aprotinin in distilled waterDW).

[0054] Subsequently, the aortic valves were incubated for 24 hours withshaking at ambient temperature in (0.05%-0.1%) (w/v) sodium dodecylsulphate (SDS) or 0.5% sodium deoxycholate in hypotonic tris buffer.They were then washed (×3) with tris buffered saline (0.15M NaCl, 0.05Mtris pH 7.6 in DW) containing protease inhibitors (0.1% w/v EDTA and 10KIU/ml Aprotinin). They were then subjected to a further wash (×3) withtris buffered saline (TBS) without protease inhibitors.

[0055] The aortic valves were then incubated for 4-6 hours at 37° C.with DNAse I (20 μg/ml) and RNAse A (1 μg/ml). After this they werewashed (×3) in TBS containing protease inhibitors. Finally, inpreparation for storage they were placed in cryoprotectant [Dulbeccomodified eagles medium (DMEM) containing 10% (v/v) foetal bovine serum(FBS) and 10% (v/v) dimethyl sulphoxide (DMSO) and cryopreserved inliquid nitrogen until they were required for implantation.

EXAMPLE 2 Porcine Patella Tendons

[0056] Porcine patella tendons were dissected and then washed in PBS.The tendons were incubated overnight (24 hours) in hypotonic Tris buffer(10 mM Tris pH 8, 0.1% ethylene diamine tetraacetate (EDTA), 10 KIUAprotinin in distilled water (DW)]. Tendons were subsequently incubatedfor a further 24 hours with shaking at ambient temperature in 0.03-0.1%w/v sodium dodecyl sulphate (SDS) or 0.5% sodium deoxycholate inhypotonic Tris buffer. They were then washed (×3) with PBS containingprotease inhibitors (0.1% EDTA and 10 KIU/ml Aprotinin).

[0057] With reference to FIG. 3A there is shown a photomicrograph offresh porcine patella tendon, stained with heamatoxylin and eosin(×400). FIG. 3B shows a photomicrograph of porcine patellar tendonfollowing de-cellularisation treatment as described above and alsostained with heamatoxylin and eosin (×400). It is apparent fromcomparing the Figures that decellularisation has been achieved whilstmaintaining the histoarchitecture of the material.

1. A method of preparing biological material for implantation comprisingthe steps of: (i) incubating the biological material with a buffersolution at a mild alkaline pH which includes active amounts of aproteolytic inhibitor, (ii) incubating the biological material with ananionic detergent at a mild alkaline pH at a concentration which issufficient to effect decellularisation but which maintains thehistoarchitecture of the biological material; (iii) washing thebiological material with a buffer solution at a mild alkaline pH bothwith and without active amounts of proteolytic inhibitors; (iv)incubating the biological material with one or more enzymes selectedfrom the group comprising DNase Type I, DNase Type II, and/or Rnase andoptionally, (v) placing the biological material in a cryoprotectantmedium.
 2. A method according to claim 1 which does not include anyadditional detergent incubation steps.
 3. A method according to eitherpreceding claim wherein the buffer is hypotonic and/or isotonic.
 4. Amethod according to either preceding claim further including the step ofcryopreserving the biological material in a cryogen such as liquidnitrogen until required.
 5. A method according to any preceding claimwherein the proteolytic inhibitor is ethylene diamine tetraacetic acid(EDTA) and Aprotinin.
 6. A method according to claim 5 wherein the EDTAis used at a concentration in the region of of 1 to 100 mM or 0.01-1.0%(w/v).
 7. A method according to either claim 5 or 6 wherein EDTA is usedat a concentration of 10 mM or 0.1% (w/v).
 8. A method according toclaim 5 wherein Aprotinin is used at a concentration range of 1-100 KIU.9. A method according to either claim 5 or 8 wherein Aprotinin is usedat 10 KIU.
 10. A method according to any preceding claim wherein themild alkaline conditions of step (i) are in the pH range of above 7.0and up to pH 10.0.
 11. A method according to claim 10 wherein the pH is8.0.
 12. A method according to any preceding claim wherein theincubation period of step (i) is for between 8 to 20 hours.
 13. A methodaccording to claim 12 wherein the incubation period is 14 hours.
 14. Amethod according to any preceding claim wherein the anionic detergent issodium dodecyl sulphate (SDS) or sodium deoxycholate.
 15. A methodaccording to claim 14 wherein SDS is used at a concentration equal to orbelow 0.1% (w/v) and equal to or above 0.03% (w/v).
 16. A methodaccording to claim 14 wherein sodium deoxycholate is used at aconcentration equal to or below 2.0% (w/v) and equal to or above 0.5%(w/v).
 17. A method according to any preceding claim wherein theincubation period of step (ii) is between 20 to 28 hours.
 18. A methodaccording to claim 17 wherein the incubation period is 24 hours.
 19. Amethod according to any preceding claim wherein the alkaline conditionsof step (ii) are in the region of are in the pH range of above 7.0 andup to pH 10.0.
 20. A method according to claim 19 wherein the pH is 8.0.21. A method according to any preceding claim wherein the washing step(iii) comprises multiple washes with tris buffered saline containingprotease inhibitors and multiple washes with tris buffered salinewithout the protease inhibitors.
 22. A method according to claim 21wherein the buffer is 0.15M NaCl, 0.05M tris in distilled water with orwithout EDTA and Aprotinin.
 23. A method according to any precedingclaim wherein the alkaline conditions of step (iii) are in the region ofare in the pH range of above 7.0 and up to pH 10.0.
 24. A methodaccording to claim 23 wherein the pH is 8.0.
 25. A method according toany preceding claim wherein the incubation of step (iv) is for 46 hoursat 37° C.
 26. A method according to any preceding claim wherein DNAse Iis used at a concentration in the range of 5-100 g/ml and RNAse A at aconcentration in the range of 0.1-10 μg/ml.
 27. A method according toany preceding claim wherein the biological tissue is prepared in step(v) for storage by placement in a cryoprotectant comprising Dulbecco'smodified eagles medium (D) containing between 10-20% (v/v) foetal bovineserum HUBS) and 5-15% (v/v) dimethyl sulphoxide (DMSO).
 28. A method ofdecellularising a tissue matrix using an anionic detergent at aconcentration sufficient to effect decellularisation but at aconcentration which maintains the histoarchitecture of the ECM, the soleanionic detergent being used in conjunction with EDTA and Aprotininprotease inhibitors.
 29. A method according to claim 28 furtherincluding any one or more of the features of claims 2 to 27.